摘要
目的 :构建云南个旧肺鳞癌YTMLC 90细胞株的cDNA文库。方法 :从培养的人肺鳞癌YTMLC 90细胞株中提取总RNA ,利用经修饰的oligo(dT)引物 (含SfiⅠB酶切位点 )合成cDNA第1链。同时 ,根据真核生物mRNA 5′端帽子结构的特点 ,利用SMART核苷酸 (含SfiⅠA酶切位点 )作为cDNA第 1链在mR NA 5′端延伸出去的模板。以此序列为引物 ,利用LD PCR(long distancePCR)合成双链cDNA。双链cDNA经SfiⅠ (ⅠA和ⅠB)酶切和过柱分级分离后 ,克隆入经SfiⅠ酶切的载体λTripIE× 2 ,经体外包装即为cDNA文库。结果 :人肺癌细胞株YTMLC 90的cDNA文库中 ,含有 1.0 1× 10 9pfu/L个重组子 ,重组率为 93.2 %。将该文库扩增后 ,克隆数可达 5 .2 4× 10 12 pfu/L ,插入cDNA的长度为 75 0～ 30 0 0bp。结论 :所构建的cDNA文库具有较好的质量 ,为进一步筛选、克隆肺癌特异性表达基因奠定了基础。
AIM: To construct the cDNA library for Yunnan Gejiu human lung cancer cell line YTMLC-90. METHODS: The total RNA was extracted from YTMLC-90 cells and the first-strand cDNA was synthesized by reverse transcription with a modified oligo(dT)primer (containing Sfi ⅠB digestion site). Simultaneously, the SMART oligonucleotide (contained Sfi I A digestion site) was utilized as a template that the first-strand of cDNA could be extended out the 5′ terminal of mRNA. The ds cDNA was amplified by LD-PCR (long-distance PCR) and then digested with SfiⅠ(ⅠA &ⅠB). After fractionation of cDNA through CHROMA SPIN column, the ds cDNA was cloned into λTripIE×2 vector which was then packaged. RESULTS: The unamplified cDNA library for human lung cancer cells consisted of 1.01×109 pfu/L independent clones in which the recombinant rate was about 93.2%. The clone number in the amplified cDNA library reached 5.24×10 12 pfu/L and the length of inserted exogenous cDNA sequence was 750-3 000 bp. CONCLUSION: The constructed cDNA library for YTMLC-90 cells has an excellent quality, which lays a solid foundation for further screening and cloning novel tissue-specific genes of human lung cancer.
出处
《细胞与分子免疫学杂志》
CAS
CSCD
北大核心
2004年第4期465-468,共4页
Chinese Journal of Cellular and Molecular Immunology
基金
国家"十五"科技攻关项目资助 (No .2 0 0 1BA70 3B1 1 )
关键词
肺癌
CDNA文库
构建
鉴定
lung cancer
cDNA library
construction
identification
